Untargeted Assessment of Tumor Fractions in Plasma for Monitoring and Prognostication from Metastatic Breast Cancer Patients Undergoing Systemic Treatment.

The aim of this study was to assess the prognostic and predictive value of an untargeted assessment of tumor fractions in the plasma of metastatic breast cancer patients and to compare circulating tumor DNA (ctDNA) with circulating tumor cells (CTC) and conventional tumor markers. In metastatic breast cancer patients (n = 29), tumor fractions in plasma were assessed using the untargeted mFAST-SeqS method from 127 serial blood samples. Resulting z-scores for the ctDNA were compared to tumor fractions established with the recently published ichorCNA algorithm and associated with the clinical outcome. We observed a close correlation between mFAST-SeqS z-scores and ichorCNA ctDNA quantifications. Patients with mFAST-SeqS z-scores above three (34.5%) showed significantly worse overall survival (p = 0.014) and progression-free survival (p = 0.018) compared to patients with lower values. Elevated z-score values were clearly associated with radiologically proven progression. The baseline CTC count, carcinoembryonic antigen (CEA), and cancer antigen (CA)15-5 had no prognostic impact on the outcome of patients in the analyzed cohort. This proof of principle study demonstrates the prognostic impact of ctDNA levels detected with mFAST-SeqS as a very fast and cost-effective means to assess the ctDNA fraction without prior knowledge of the genetic landscape of the tumor. Furthermore, mFAST-SeqS-based ctDNA levels provided an early means of measuring treatment response.

Multigene methylation analysis of enriched circulating tumor cells associates with poor progression-free survival in metastatic breast cancer patients.

Blood-based biomarkers such as circulating tumor cells (CTCs) provide dynamic real-time assessment of molecular tumor characteristics beyond the primary tumor. The aim of this study was to evaluate the feasibility of a size-based microfilter to assess multigene methylation analysis of enriched CTCs in a prospective proof-of principle study. We examined the quantitative methylation status of nine genes (AKR1B1, BMP6, CST6, HOXB4, HIST1H3C, ITIH5, NEUROD1, RASSF1, SOX17) in enriched CTCs from metastatic breast cancer patients. Feasibility and clinical performance testing were assessed in a test set consisting of 37 patients and 25 healthy controls. With established cut-off values from the healthy control group, methylation of enriched CTCs was detected in at least one gene in 18/37 patients (48.6%), while 97.8% of all control samples were unmethylated. Patients with CTCs unmethylated for CST6, ITIH5, or RASSF1 showed significantly longer PFS compared to patients with corresponding enriched methylated CTCs. This proof-of-principle study shows the feasibility of a size-based microfilter to enrich and analyze multigene methylation profile of CTCs from metastatic breast cancer patients. For the first time, we report that multigene methylation analysis of enriched CTCs provides prognostic information in metastatic breast cancer patients.

Microfilter-Based Capture and Release of Viable Circulating Tumor Cells.

Microfilters with slot-pore geometry can be used for size-based capture of circulating tumor cells (CTC) from the blood of cancer patients. The slot pore geometry reduces the shear stress that the cells would typically experience during filtration process and allows the cells to remain viable. The microfilter provides a platform capable of high CTC capture efficiency; however, the release of these cells from the filter following capture is nontrivial, possibly due to the strong nonspecific electrostatic adhesion of CTC to the microfilter surface. Techniques such as reverse flow or cell scraping result in recovery of only a small percentage of captured cells. We describe, in detail, a protocol for novel application of thermo-responsive polymer poly(N-iso-propylacrylamide) (PIPAAm) to release viable CTCs from microfilters with slot pores. Following fabrication of the microfilter, a coating of PIPAAm is applied to the surface to exploit its thermoresponsive interfacial properties to release the cells. Typically, cancer patient's blood is filtered at room temperature (below 32 °C) when PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture medium or a buffer maintained at 37 °C, which renders PIPAAm hydrophobic, allowing subsequent release of the electrostatically bound cells with high efficiency. Using this method, viable CTC captured directly from cancer patients' blood can be subjected to downstream off-chip culture, analyses, and characterization.

Interactions between Adipocytes and Breast Cancer Cells Stimulate Cytokine Production and Drive Src/Sox2/miR-302b-Mediated Malignant Progression.

Consequences of the obesity epidemic on cancer morbidity and mortality are not fully appreciated. Obesity is a risk factor for many cancers, but the mechanisms by which it contributes to cancer development and patient outcome have yet to be fully elucidated. Here, we examined the effects of coculturing human-derived adipocytes with established and primary breast cancer cells on tumorigenic potential. We found that the interaction between adipocytes and cancer cells increased the secretion of proinflammatory cytokines. Prolonged culture of cancer cells with adipocytes or cytokines increased the proportion of mammosphere-forming cells and of cells expressing stem-like markers in vitro. Furthermore, contact with immature adipocytes increased the abundance of cancer cells with tumor-forming and metastatic potential in vivo. Mechanistic investigations demonstrated that cancer cells cultured with immature adipocytes or cytokines activated Src, thus promoting Sox2, c-Myc, and Nanog upregulation. Moreover, Sox2-dependent induction of miR-302b further stimulated cMYC and SOX2 expression and potentiated the cytokine-induced cancer stem cell-like properties. Finally, we found that Src inhibitors decreased cytokine production after coculture, indicating that Src is not only activated by adipocyte or cytokine exposures, but is also required to sustain cytokine induction. These data support a model in which cancer cell invasion into local fat would establish feed-forward loops to activate Src, maintain proinflammatory cytokine production, and increase tumor-initiating cell abundance and metastatic progression. Collectively, our findings reveal new insights underlying increased breast cancer mortality in obese individuals and provide a novel preclinical rationale to test the efficacy of Src inhibitors for breast cancer treatment.

A surface acoustic wave biosensor for interrogation of single tumour cells in microcavities.

In this study, biological cells are sensed and characterized with surface acoustic wave (SAW) devices utilising microcavities. After tumour cells in media are transported to and trapped in microcavities, the proposed platform uses SAW interaction between the substrate and the cells to extract their mechanical stiffness based on the ultrasound velocity. Finite element method (FEM) analysis and experimental results show that output phase information is an indicator of the stiffness modulus of the trapped cells. Small populations of various types of cells such as MCF7, MDA-MB-231, SKBR3, and JJ012 were characterized and characteristic moduli were estimated for each cell population. Results show that high frequency stiffness modulus is a possible biomarker for aggressiveness of the tumour and that microcavity coupled SAW devices are a good candidate for non-invasive interrogation of single cells.

Thermoresponsive release of viable microfiltrated Circulating Tumor Cells (CTCs) for precision medicine applications.

Stimulus responsive release of Circulating Tumor Cells (CTCs), with high recovery rates from their capture platform, is highly desirable for off-chip analyses. Here, we present a temperature responsive polymer coating method to achieve both release as well as culture of viable CTCs captured from patient blood samples.

Identification of Cancer-Associated Fibroblasts in Circulating Blood from Patients with Metastatic Breast Cancer.

Metastasis is facilitated by cancer-associated fibroblasts (CAF) in the tumor microenvironment through mechanisms yet to be elucidated. In this study, we used a size-based microfilter technology developed by our group to examine whether circulating CAF identified by FAP and α-SMA co-expression (cCAF) could be distinguished in the peripheral blood of patients with metastatic breast cancer. In a pilot study of patients with breast cancer, we detected the presence of cCAFs in 30/34 (88%) patients with metastatic disease (MET group) and in 3/13 (23%) patients with localized breast cancer (LOC group) with long-term disease-free survival. No cCAFs as defined were detected in healthy donors. Further, both cCAF and circulating tumor cells (CTC) were significantly greater in the MET group compared with the LOC group. Thus, the presence of cCAF was associated with clinical metastasis, suggesting that cCAF may complement CTC as a clinically relevant biomarker in metastatic breast cancer.

Highly scalable, uniform, and sensitive biosensors based on top-down indium oxide nanoribbons and electronic enzyme-linked immunosorbent assay.

Nanostructure field-effect transistor (FET) biosensors have shown great promise for ultra sensitive biomolecular detection. Top-down assembly of these sensors increases scalability and device uniformity but faces fabrication challenges in achieving the small dimensions needed for sensitivity. We report top-down fabricated indium oxide (In2O3) nanoribbon FET biosensors using highly scalable radio frequency (RF) sputtering to create uniform channel thicknesses ranging from 50 to 10 nm. We combine this scalable sensing platform with amplification from electronic enzyme-linked immunosorbent assay (ELISA) to achieve high sensitivity to target analytes such as streptavidin and human immunodeficiency virus type 1 (HIV-1) p24 proteins. Our approach circumvents Debye screening in ionic solutions and detects p24 protein at 20 fg/mL (about 250 viruses/mL or about 3 orders of magnitude lower than commercial ELISA) with a 35% conduction change in human serum. The In2O3 nanoribbon biosensors have 100% device yield and use a simple 2 mask photolithography process. The electrical properties of 50 In2O3 nanoribbon FETs showed good uniformity in on-state current, on/off current ratio, mobility, and threshold voltage. In addition, the sensors show excellent pH sensitivity over a broad range (pH 4 to 9) as well as over the physiological-related pH range (pH 6.8 to 8.2). With the demonstrated sensitivity, scalability, and uniformity, the In2O3 nanoribbon sensor platform makes great progress toward clinical testing, such as for early diagnosis of acquired immunodeficiency syndrome (AIDS).

Circulating tumor cell counts are prognostic of overall survival in SWOG S0421: a phase III trial of docetaxel with or without atrasentan for metastatic castration-resistant prostate cancer.

PURPOSE: Circulating tumor cell (CTC) enumeration has not been prospectively validated in standard first-line docetaxel treatment for metastatic castration-resistant prostate cancer. We assessed the prognostic value of CTCs for overall survival (OS) and disease response in S0421, a phase III trial of docetaxel plus prednisone with or without atrasentan. PATIENTS AND METHODS: CTCs were enumerated at baseline (day 0) and before cycle two (day 21) using CellSearch. Baseline counts and changes in counts from day 0 to 21 were evaluated for association with OS, prostate-specific antigen (PSA), and RECIST response using Cox regression as well as receiver operator characteristic (ROC) curves, integrated discrimination improvement (IDI) analysis, and regression trees. RESULTS: Median day-0 CTC count was five cells per 7.5 mL, and CTCs < versus ≥ five per 7.5 mL were significantly associated with baseline PSA, bone pain, liver disease, hemoglobin, alkaline phosphatase, and subsequent PSA and RECIST response. Median OS was 26 months for < five versus 13 months for ≥ five CTCs per 7.5 mL at day 0 (hazard ratio [HR], 2.74 [adjusting for covariates]). ROC curves had higher areas under the curve for day-0 CTCs than for PSA, and IDI analysis showed that adding day-0 CTCs to baseline PSA and other covariates increased predictive accuracy for survival by 8% to 10%. Regression trees yielded new prognostic subgroups, and rising CTC count from day 0 to 21 was associated with shorter OS (HR, 2.55). CONCLUSION: These data validate the prognostic utility of CTC enumeration in a large docetaxel-based prospective cohort. Baseline CTC counts were prognostic, and rising CTCs at 3 weeks heralded significantly worse OS, potentially serving as an early metric to help redirect and optimize therapy in this clinical setting.

Genetic and epigenetic analysis of putative breast cancer stem cell models.

BACKGROUND: Cancer stem cell model hypothesizes existence of a small proportion of tumor cells capable of sustaining tumor formation, self-renewal and differentiation. In breast cancer, these cells were found to be associated with CD44⁺CD24-low and ALDH⁺ phenotype. Our study was performed to evaluate the suitability of current approaches for breast cancer stem cell analyses to evaluate heterogeneity of breast cancer cells through their extensive genetic and epigenetic characterization. METHODS: Breast cancer cell lines MCF7 and SUM159 were cultured in adherent conditions and as mammospheres. Flow cytometry sorting for CD44, CD24 and ALDH was performed. Sorted and unsorted populations, mammospheres and adherent cell cultures were subjected to DNA profiling by array CGH and methylation profiling by Epitect Methyl qPCR array. Methylation status of selected genes was further evaluated by pyrosequencing. Functional impact of methylation was evaluated by mRNA analysis for selected genes. RESULTS: Array CGH did not reveal any genomic differences. In contrast, putative breast cancer stem cells showed altered methylation levels of several genes compared to parental tumor cells. CONCLUSIONS: Our results underpin the hypothesis that epigenetic mechanisms seem to play a major role in the regulation of CSCs. However, it is also clear that more efficient methods for CSC enrichment are needed. This work underscores requirement of additional approaches to reveal heterogeneity within breast cancer.

A novel precision-engineered microfiltration device for capture and characterisation of bladder cancer cells in urine.

BACKGROUND: Sensitivity of standard urine cytology for detecting urothelial carcinoma of the bladder (UCB) is low, attributable largely to its inability to process entire samples, paucicellularity and presence of background cells. OBJECTIVE: Evaluate performance and practical applicability of a novel portable microfiltration device for capture, enumeration and characterisation of exfoliated tumour cells in urine, and compare it with standard urine cytology for UCB detection. METHODS: A total of 54 urine and bladder wash samples from patients undergoing surveillance for UCB were prospectively evaluated by standard and microfilter-based urine cytology. Head-to-head comparison of quality and performance metrics, and cost effectiveness was conducted for both methodologies. RESULTS: Five samples were paucicellular by standard cytology; no samples processed by microfilter cytology were paucicellular. Standard cytology had 33.3% more samples with background cells that limited evaluation (p<0.001). Microfilter cytology was more concordant (κ=50.4%) than standard cytology (κ=33.5%) with true UCB diagnosis. Sensitivity, specificity and accuracy were higher for microfilter cytology compared to standard cytology (53.3%/100%/79.2% versus 40%/95.8%/69.9%, respectively). Microfilter-captured cells were amenable to downstream on-chip molecular analyses. A 40 ml sample was processed in under 4 min by microfilter cytology compared to 5.5 min by standard cytology. Median microfilter cytology processing and set-up costs were approximately 63% cheaper and 80 times lower than standard cytology, respectively. CONCLUSIONS: The microfiltration device represents a novel non-invasive UCB detection system that is economical, rapid, versatile and has potentially better quality and performance metrics than routine urine cytology, the current standard-of-care.

Combination of molecular alterations and smoking intensity predicts bladder cancer outcome: a report from the Los Angeles Cancer Surveillance Program.

BACKGROUND: Traditional single-marker and multimarker molecular profiling approaches in bladder cancer do not account for major risk factors and their influence on clinical outcome. This study examined the prognostic value of molecular alterations across all disease stages after accounting for clinicopathological factors and smoking, the most common risk factor for bladder cancer in the developed world, in a population-based cohort. METHODS: Primary bladder tumors from 212 cancer registry patients (median follow-up, 13.2 years) were immunohistochemically profiled for Bax, caspase-3, apoptotic protease-activating factor 1 (Apaf-1), Bcl-2, p53, p21, cyclooxygenase-2, vascular endothelial growth factor, and E-cadherin alterations. "Smoking intensity" quantified the impact of duration and daily frequency of smoking. RESULTS: Age, pathological stage, surgical modality, and adjuvant therapy administration were significantly associated with survival. Increasing smoking intensity was independently associated with worse outcome (P < .001). Apaf-1, E-cadherin, and p53 were prognostic for outcome (P = .005, .014, and .032, respectively); E-cadherin remained prognostic following multivariable analysis (P = .040). Combined alterations in all 9 biomarkers were prognostic by univariable (P < .001) and multivariable (P = .006) analysis. A multivariable model that included all 9 biomarkers and smoking intensity had greater accuracy in predicting prognosis than models composed of standard clinicopathological covariates without or with smoking intensity (P < .001 and P = .018, respectively). CONCLUSIONS: Apaf-1, E-cadherin, and p53 alterations individually predicted survival in bladder cancer patients. Increasing number of biomarker alterations was significantly associated with worsening survival, although markers comprising the panel were not necessarily prognostic individually. Predictive value of the 9-biomarker panel with smoking intensity was significantly higher than that of routine clinicopathological parameters alone.

Progress in circulating tumor cell capture and analysis: implications for cancer management.

The hematogenous dissemination of cancer and development of distant metastases is the cause of nearly all cancer deaths. Detection of circulating tumor cells (CTCs) as a surrogate biomarker of metastases has gained increasing interest. There is accumulating evidence on development of novel technologies for CTC detection, their prognostic relevance and their use in therapeutic response monitoring. Many clinical trials in the early and metastatic cancer setting, particularly in breast cancer, are including CTCs in their translational research programs and as secondary end points. We summarize the progress of detection methods in the context of their clinical importance and speculate on the possibilities of wider implementation of CTCs as a diagnostic oncology tool, the likelihood that CTCs will be used as a useful biomarker, especially to monitor therapeutic response, and what may be expected from the future improvements in technologies.

Portable filter-based microdevice for detection and characterization of circulating tumor cells.

PURPOSE: Sensitive detection and characterization of circulating tumor cells (CTC) could revolutionize the approach to patients with early-stage and metastatic cancer. The current methodologies have significant limitations, including limited capture efficiency and ability to characterize captured cells. Here, we report the development of a novel parylene membrane filter-based portable microdevice for size-based isolation with high recovery rate and direct on-chip characterization of captured CTC from human peripheral blood. EXPERIMENTAL DESIGN: We evaluated the sensitivity and efficiency of CTC capture in a model system using blood samples from healthy donors spiked with tumor cell lines. Fifty-nine model system samples were tested to determine the recovery rate of the microdevice. Moreover, 10 model system samples and 57 blood samples from cancer patients were subjected to both membrane microfilter device and CellSearch platform enumeration for direct comparison. RESULTS: Using the model system, the microdevice achieved >90% recovery with probability of 95% recovering at least one cell when five are seeded in 7.5 mL of blood. CTCs were identified in 51 of 57 patients using the microdevice, compared with only 26 patients with the CellSearch method. When CTCs were detected by both methods, greater numbers were recovered by the microfilter device in all but five patients. CONCLUSIONS: This filter-based microdevice is both a capture and analysis platform, capable of multiplexed imaging and genetic analysis. The microdevice presented here has the potential to enable routine CTC analysis in the clinical setting for the effective management of cancer patients.

Predicting recurrence and progression of noninvasive papillary bladder cancer at initial presentation based on quantitative gene expression profiles.

BACKGROUND: Currently, tumor grade is the best predictor of outcome at first presentation of noninvasive papillary (Ta) bladder cancer. However, reliable predictors of Ta tumor recurrence and progression for individual patients, which could optimize treatment and follow-up schedules based on specific tumor biology, are yet to be identified. OBJECTIVE: To identify genes predictive for recurrence and progression in Ta bladder cancer at first presentation using a quantitative, pathway-specific approach. DESIGN, SETTING, AND PARTICIPANTS: Retrospective study of patients with Ta G2/3 bladder tumors at initial presentation with three distinct clinical outcomes: absence of recurrence (n=16), recurrence without progression (n=16), and progression to carcinoma in situ or invasive disease (n=16). MEASUREMENTS: Expressions of 24 genes that feature in relevant pathways that are deregulated in bladder cancer were quantified by real-time polymerase chain reaction on tumor biopsies from the patients at initial presentation. RESULTS AND LIMITATIONS: CCND3 (p=0.003) and HRAS (p=0.01) were predictive for recurrence by univariate analysis. In a multivariable model based on CCND3 expression, sensitivity and specificity for recurrence were 97% and 63%, respectively. HRAS (p<0.001), E2F1 (p=0.017), BIRC5/Survivin (p=0.038), and VEGFR2 (p=0.047) were predictive for progression by univariate analysis. Multivariable analysis based on HRAS, VEGFR2, and VEGF identified progression with 81% sensitivity and 94% specificity. Since this is a small retrospective study using medium-throughput profiling, larger confirmatory studies are needed. CONCLUSIONS: Gene expression profiling across relevant cancer pathways appears to be a promising approach for Ta bladder tumor outcome prediction at initial diagnosis. These results could help differentiate between patients who need aggressive versus expectant management.

Generation of a concise gene panel for outcome prediction in urinary bladder cancer.

PURPOSE: This study sought to determine if alterations in molecular pathways could supplement TNM staging to more accurately predict clinical outcome in patients with urothelial carcinoma (UC). PATIENTS AND METHODS: Expressions of 69 genes involved in known cancer pathways were quantified on bladder specimens from 58 patients with UC (stages Ta-T4) and five normal urothelium controls. All tumor transcript values beyond two standard deviations from the normal mean expression were designated as over- or underexpressed. Univariate and multivariable analyses were conducted to obtain a predictive expression signature. A published external data set was used to confirm the potential of the prognostic gene panels. RESULTS: In univariate analysis, six genes were significantly associated with time to recurrence, and 10 with overall survival. Recursive partitioning identified three genes as significant determinants for recurrence, and three for overall survival. Of all genes identified by either univariate or partitioning analysis, four were found to significantly predict both recurrence and survival (JUN, MAP2K6, STAT3, and ICAM1); overexpression was associated with worse outcome. Comparing the favorable (low or normal) expression of > or = three of four versus < or = two of four of these oncogenes showed 5-year recurrence probability of 41% versus 88%, respectively (P < .001), and 5-year overall survival probability of 61% versus 5%, respectively (P < .001). The prognostic potential of this four-gene panel was confirmed in a large independent external cohort (disease-specific survival, P = .039). CONCLUSION: We have documented the generation of a concise, biologically relevant four-gene panel that significantly predicts recurrence and survival and may also identify potential therapeutic targets for UC.

Unmethylated E-cadherin gene expression is significantly associated with metastatic human prostate cancer cells in bone.

BACKGROUND: The concurrent determination of methylation status of E-cadherin gene and E-cadherin protein expression remains scant in metastatic prostate cancer cells in bone, the most prevalent site for metastatic growth. Therefore, the study was undertaken to ascertain the methylation status of E-cadherin gene, a most frequent and known epigenetic mechanism of its regulation, and the protein expression in prostate tissue biopsy specimen. METHODS: The methylation of E-cadherin gene was determined by methylation specific-PCR and the protein expression by immunohistochemical method in the consecutive sections of each prostate tissue biopsy specimen. RESULTS: The unmethylated E-cadherin gene and homogeneous E-cadherin protein expression was significantly associated with BPH as compared to the primary prostate carcinoma (Fisher's Exact P < 0.001). A significant association was observed between the concurrent methylated gene and markedly reduced expression of the protein in the primary prostate cancer cells as compared to the BPH cells, suggesting methylation-dependent regulation of the gene expression in these cases. In contrast to the primary cancer, a highly significant increase in the frequency of metastatic prostate cancer cells in bone exhibited the concurrent expression of unmethylated gene and homogeneous protein (Fisher's Exact P < 0.001). CONCLUSIONS: The study clearly demonstrated a significant association of the concurrent expression of unmethylated E-cadherin gene and E-cadherin protein with metastatic prostate cancer cells in bone, and that its expression may have a role in the intercellular adhesion in the formation of metastatic lesions in bone.

p53 gene and protein status: the role of p53 alterations in predicting outcome in patients with bladder cancer.

PURPOSE: The p53 gene status (mutation) and protein alterations (nuclear accumulation detectable by immunohistochemistry; p53 protein status) are associated with bladder cancer progression. Substantial discordance is documented between the p53 protein and gene status, yet no studies have examined the relationship between the gene-protein status and clinical outcome. This study evaluated the clinical relationship of the p53 gene and protein statuses. MATERIALS AND METHODS: The complete coding region of the p53 gene was queried using DNA from paraffin-embedded tissues and employing a p53 gene-sequencing chip. We compared p53 gene status, mutation site, and protein status with time to recurrence. RESULTS: The p53 gene and protein statuses show significant concordance, yet 35% of cases showed discordance. Exon 5 mutations demonstrated a wild-type protein status in 18 of 22 samples. Both the p53 gene and protein statuses were significantly associated with stage and clinical outcome. Specific mutation sites were associated with clinical outcome; tumors with exon 5 mutations showed the same outcome as those with the wild-type gene. Combining the p53 gene and protein statuses stratifies patients into three distinct groups, based on recurrence-free intervals: patients showing the best outcome (wild-type gene and unaltered protein), an intermediate outcome (either a mutated gene or an altered protein) and the worst outcome (a mutated gene and an altered protein). CONCLUSION: We show that evaluation of both the p53 gene and protein statuses provides information in assessing the clinical recurrence risk in bladder cancer and that the specific mutation site may be important in assessing recurrence risk. These findings may substantially impact the assessment of p53 alterations and the management of bladder cancer.

The role of deoxyribonucleic acid methylation in development, diagnosis, and prognosis of bladder cancer.

Alterations in global levels and regional patterns of deoxyribonucleic acid methylation are among the earliest and most common events known to occur in human cancer. The mutational and epigenetic effects of this covalent deoxyribonucleic acid modification to the development of bladder cancer are well recognized. The contribution of aberrant methylation to mutational hot spots located within genes, transcriptional silencing, and chromosomal instability is reviewed in the context of its relevance to bladder carcinogenesis. Understanding how such processes evolve during the progression of bladder cancer is essential for using these molecular changes in the clinical setting. The recent development of sensitive and specific techniques for quantifying methylation changes in urine specimens and bodily fluids underscores the potential use of this molecular marker for early detection and surveillance of bladder cancer. Further refinement of these molecular biological techniques holds much promise for the use of methylation markers for bladder cancer diagnosis, risk stratification, and disease prognostication.